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PubMed1984

Erythrocytic pyruvate kinase deficiency and hemolytic anemia inherited as a dominant trait.

A nonspherocytic hemolytic anemia, associated with a pyruvate kinase (PK) deficiency apparently inherited as a dominant trait has been identified in a family. In the affected members, the residual PK activities were 20% that of normal controls, an unusually low level for heterozygous subjects. The anemia was mild except in the proband, a 2-year-old boy who suffered from a severe anemia. The PKs of the proband and of his both parents have been characterized as kinetically and electrophoretically normal enzymes. Immunoprecipitation tests indicated a large amount of L-type inactive protein in the proband and his father. Moreover, an M2 type PK was present in the father's hemolysate, suggesting the existence of a compensatory process that derepressed the corresponding structural gene. We suggest that the presence of one or more mutated subunits in the tetrameric forms of L-type PK leads to the inactivation of these tetramers. This hypothesis accounts for the low residual activity in affected heterozygous members of this family. The severity of the hematological symptoms in the proband, in comparison with the mild hemolysis observed in the other heterozygous members of the family points to the existence of a large spectrum of pathologic expression for an identical PK defect present in a family.

Adult↗
PubMed1993

[Further study on the value of M2-type pyruvate kinase in the diagnosis of primary hepatocarcinoma].

Based on the pilot study of plasma M2-type pyruvate kinase (M2-PyK) in the diagnosis of primary hepatocarcinoma, some improvements in methodology were made. M2-PyK was purified directly from human placenta, and anti-human M2-PyK antibody was raised in rabbits. The purified antibody IgG was degraded into Fab' fragment and cross-linked with horseradish peroxidase (HRP) to prepare Fab'-HRP conjugate. By using sandwich Fab'-ELISA, M2-PyK in the plasma was determined in 119 healthy adults, all gave values less than 2.35 ng/ml, with 80 percent less than 1.2 ng/ml, and the medium was 0.68ng/ml. There was no significant difference between male and female subjects. The plasma M2-PyK in 32 cases of primary hepatocarcinoma was markedly increased with a range of 1.31-30.13ng/ml or 7.06 +/- 5.25 ng/ml, which was more than 10 fold of the normal medium value. If 2.35 ng/ml was taken as the upper limit of the normal range, the positive rate was up to 90.6%. Seven cases of hepatocarcinoma with AFP less than 50 ng/ml, M2-PyK was positive. The result was the same as that in our previous report. Other primary malignant tumors of the liver origin were also positive, whereas the benign tumors of the liver were negative. In 53 cases of the carcinomas in gastro-intestinal tract, whether the carcinomas were metastasized to the liver or not, a positive rate of 40.6-52.3% was shown which was higher than that in the cases of colorectal carcinoma. Hence, this method was not specific for the diagnosis of primary hepatocarcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMedDate not supplied

Pre-analytical problems in the measurement of tumor type pyruvate kinase (tumor M2-PK).

INTRODUCTION: Tumor M2-PK is an isoform of the glycolytic enzyme pyruvate kinase. This isoform exists in an active tetrameric and less active dimeric form. The dimeric form is expressed by various tumor cells and can be measured in blood by a specific ELISA. MATERIALS AND METHODS: We included 500 healthy persons, 20 patients with an acute rheumatic disease (high CRP) and 30 patients with a nephropathy and proteinuria with more than 20 mg/dl. We measured Tumor M2-PK in each of these groups in different specimens of EDTA-plasma, serum, heparin-, citrate-, fluoride- and oxalate-plasma. RESULTS: We found different concentrations of Tumor M2-PK in healthy persons depending on the kind of specimen. The normal range of Tumor M2-PK concentration was higher in the serum than in the plasma, with 35 U/l compared to 15 U/l. In haemolytic material we found concentrations up to 80 U/l in healthy persons. Lipaemic or icteric serum material showed a higher concentration of Tumor M2-PK as well. In patients with a triglyceride concentration higher than 300 mg/dl, the normal range for Tumor M2-PK was measured between 30-50 U/l. Similar results were found in patients with icteric serum. Patients with an active rheumatic disease and elevated CRP concentration showed a Tumor M2-PK concentration between 40-60 U/l. Patients with a nephropathy and proteinuria over 20 mg/dl had an elevated Tumor M2-PK concentration from 25-60 U/l. CONCLUSION: We conclude that different materials seem to be suitable for the measurement of Tumor M2-PK concentration as long as attention is paid to different normal ranges. Haemolytic material should not be used as it offers false-positive results. Some diseases such as rheumatic disease, hyperlipidaemia and nephropathy have an influence on Tumor M2-PK concentration that should not be neglected.

Biomarkers, Tumor↗
PubMed1985

Comparative kinetic studies of Mn2+-activated and fructose-1,6-P-modified Mg2+-activated pyruvate kinase from Concholepas concholepas.

Initial velocity and product inhibition studies of Mn2+-activated and FDP-modified Mg2+-activated pyruvate kinase from Concholepas concholepas, were performed. Evidence is presented to show that the Mn2+-enzyme catalyzes an ordered sequential mechanism, with ADP being the first substrate and pyruvate the last product. The results presented are consistent with a random combination of reactants with the FDP-modified Mg2+-activated enzyme and the formation of the dead-end complexes enzyme ADP-ATP and enzyme-PEP-ATP.

Animals↗
PubMed1996

Alternative splicing of the pyruvate kinase M gene in a minigene system.

The M1-type and M2-type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M2 type, which occurs in most tissues, whereas the M1 type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M2-expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse-transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh-84 cells, which express only the M2 type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M1 or M2 types. The 5' splice site but not the 3' splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh-84 cells. Mutation of the 5' splice site sequence of exon 9 to that of exon 10 and vice versa did not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. A 9-10 fusion exon (constructed by substitution of 68 bases of the 3' portion of exon 9 and 33 bases of the 5' portion of intron 9 for the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non M1-expressing cells due to the weak signal of its 5' splice site and that, although the 5' splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' portion of exon 9 and the 5' portion of intron 9 and/or the corresponding regions of exon 10 and intron 10.

Alternative Splicing↗
PubMed2002

Metabolic flux responses to pyruvate kinase knockout in Escherichia coli.

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

Allosteric Regulation↗
PubMed2004

Pyruvate kinase activity in the placentas of women living in polluted and unpolluted environments.

BACKGROUND: It was shown earlier that in women living in a polluted environment, the proportion of hypotrophic newborns was greater than in the general population. The placentas of these women show significant histological changes. The authors decided to check whether the activity of pyruvate kinase (PK), a key enzyme in the third stage of glycolysis, changes similarly. MATERIAL/METHODS: The study was conducted on placentas collected from women who had lived in a polluted environment (Groups I and II) and those who had inhabited an unpolluted area (Group III) while pregnant. RESULTS: Total PK activity and the specific activities of its isoenzymes were measured according to the Bücher and Pfleiderer method. The isolation of PK isoenzymes was carried out following the previously described procedure. Total PK activity was lower in the placentas of women of Groups I and II than in those of Group III. The presence of two PK isoenzymes, M2 and L, was demonstrated. The M2 and L isoenzymes from the placentas of Groups I and II had a lower specific activity than those of Group III. Only one form of the M2 isoenzyme and the L dephospho-isoenzyme in the placentas from Groups I and II was observed, while in placentas of Group III women both forms of the M2 isoenzyme and the L phospho-isoenzyme were noted. CONCLUSIONS: It is possible that the presence of the two PK isoenzymes in the placenta ensures the production of a sufficient amount of pyruvate.

Air Pollution↗
PubMed1991

The monomer of pyruvate kinase, subtype M1, is both a kinase and a cytosolic thyroid hormone binding protein.

Using a T7 expression system, the monomer of rat pituitary pyruvate kinase, subtype M1 (PKM1), was overexpressed in Escherichia coli and purified to homogeneity. The monomeric p58-M1 has intrinsic enzymatic activity with a Vmax of 79 +/- 20 units/mg and Km's for ADP and PEP of 1.43 +/- 0.76 and 0.14 +/- 0.07 mM, respectively. The monomer binds 3,3',5-triiodo-L-thyronine (T3) with Ka = 1.5 x 10(7) M-1. The order of analog specificity is L-T3 greater than L-thyroxine greater than D-T3 greater than 3'-isopropyl-3,5-diiodo-L-thyronine greater than or equal to 3',5',3-triiodo-L-thyronine. In contrast, tetrameric PKM1 lacks T3 binding activity. The kinase activity of p58-M1 is inhibited by T3 and its analogs in a concentration-dependent manner with the order of inhibitory activity similar to that of binding activity. This inhibition, however, is reversed by the addition of fructose 1,6-bisphosphate. p58-M1 is the second PK isoenzyme monomer to be identified as having thyroid hormone binding activity.

Animals↗
PubMed1981

Studies on pyruvate kinase deficiency, pyrimidine 5-'nucleotidase deficiency and adenosine deaminase overproduction.

Using recently established ICSH recommended methods, red cell pyruvate kinases (PK) of 20 patients with PK deficiency were characterized and 7 new PK variants were found. Analysis of partially purified red cell pyrimidine 5'-nucleotidase (P5N) from a patient with P5N deficiency provided the evidence for a structural alteration of the enzyme protein. Red cell adenosine deaminase (ADA) from a patient with 40-fold increase in ADA activity associated with hemolytic anemia was purified and compared with that from normal subjects. It is most conceivable that the increased ADA activity represents increased amount of structurally normal enzyme.

5'-Nucleotidase↗
PubMed1980

Pyruvate kinase hyperactivity genetically determined metabolic consequences and molecular characterization.

Four people from the same family with red cell pyruvate kinase (PK) hyperactivity are described. These people displayed low 2,3-diphosphoglycerate (2,3-DPG) and high adenosine triphosphate (ATP) levels. In vitro metabolism studies of their red cells showed the relationship between the PK activity, low 2,3-DPG, and high ATP levels. The PK electrophoretic pattern of these subjects was abnormal by the presence of several additional bands; one of them migrated like PKM2. PKR from these people was thermounstable and M2-like PK was identical to PKM2 for immunologic reactivity and KO, 5s for phosphoenolpyruvate.

Adenosine Triphosphate↗
PubMed1986

Frequency of glucose-6-phosphate dehydrogenase, pyruvate kinase and hexokinase deficiency in the Saudi population.

The frequencies of glucose-6-phosphate dehydrogenase (G-6-PD), pyruvate kinase (PK) and hexokinase (HK) deficiency were determined in different regions of Saudi Arabia. G-6-PD deficiency was found to range from 0.045 to 0.220 for the male and 0.020 to 0.125 for the female population. The highest frequencies were found to exist in the regions which are endemic to malarial parasite and have high frequencies of sickle cell and thalassaemia genes. Partial deficiencies of PK and HK were encountered in each region, however, no case of complete deficiency of these enzymes was identified. Further investigations are in progress to determine the clinical manifestations of enzyme deficiencies in the Saudi population.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1997

Recombinant pyruvate kinase type I from Escherichia coli: overproduction and revised C-terminus of the polypeptide.

The gene encoding pyruvate kinase type I (PKI) of Escherichia coli was amplified by PCR, cloned and sequenced. The gene product was overexpressed in E. coli, using an inducible T7 RNA polymerase-based expression system. The transformed cells contained sixtyfold the enzyme activity of the reference cells and enabled the purification of 30 mg of highly active PKI from 1 liter of culture. The gene sequence was determined and found to be different from the one previously reported, i.e., the T nucleotide at position 1351 was missing. This resulted in a downstream shift of the stop codon, thus the deduced polypeptide was 470 amino acids long instead of 462. In addition the twelve C-terminal amino acids of the former sequence were changed.

Amino Acid Sequence↗
PubMed1985

Effect of Nitrate and Ammonium Nutrition of Nonnodulated Phaseolus vulgaris L. on Phosphoenolpyruvate Carboxylase and Pyruvate Kinase Activity.

Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with 3.5 or 10 millimolar N in either the form of NO(3) (-) or NH(4) (+), after being grown on N-free nutrient solution for 8 days. The pH of the nutrient solutions was either 6 or 4. The cell sap pH and the extractable activities of phosphoenolpyruvate carboxylase and of pyruvate kinase from roots and primary leaves were measured over several days.The extractable activity of phosphoenolpyruvate carboxylase (based on soluble protein) from primary leaves increased with NO(3) (-) nutrition, whereas with NH(4) (+) nutrition and on N-free nutrient solution the activity remained at a low level. Phosphoenopyruvate carboxylase activity from the roots of NH(4) (+)-fed plants at pH 4 was finally somewhat higher than from the roots of plants grown on NO(3) (-) at the same pH. There was no difference in activity from the root between the N treatments when pH in the nutrient solutions was 6. The extractable activity of pyruvate kinase from roots and primary leaves seemed not to be influenced by the N nutrition of the plants.The results are discussed in relation to the physiological function of both enzymes with special regard to the postulated functions of phosphoenolpyruvate carboxylase in C3 plants as an anaplerotic enzyme and as part of a cellular pH stat.

Journal Article↗
PubMed1985

Pyruvate kinase isozymes and dedifferentiation of (neuro-)ectodermal tumors.

This paper describes the isozyme composition and regulatory properties of pyruvate kinase (PK) from well-differentiated (DMTC) and undifferentiated (AMTC) medullary thyroid carcinomas of the rat. These tumors were chosen as an animal model for human (neuro-)entodermal neoplasms differing in their degree of differentiation. The results were compared with human medullary thyroid carcinomas (MTC) and phaeochromocytomas. AMTC were characterized by increased PK activity, a higher apparent S0.5 for phosphoenolpyruvate, enhanced inhibition by alanine, presence of predominantly M2-type isozyme and loss of M1-type-containing hybrids as compared to DMTC. The alterations in PK isozyme expression and hence kinetic behaviour could not be demonstrated in human MTC or phaeochromocytomas due to the apparently well-differentiated nature of these tumors and the presence of M2-type isozymes. These results are discussed with reference to the nature and significance of PK isozyme shifts found in other tumors. It is suggested that the determination of PK isozyme composition might prove useful in the diagnosis of nueroectodermal neoplasms originating from tissues not primarily expressing M2-type isozyme(s).

Animals↗
PubMedDate not supplied

Tumor M2-pyruvate kinase: a promising tumor marker in the diagnosis of gastro-intestinal cancer.

The diagnostic value of pyruvate-kinase type tumor M2 (Tumor M2-PK) has been investigated in different tumors, and showed interesting results in cases of renal cancer, pancreatic cancer, lung cancer and some cases of gastric cancer. In this study we investigated EDTA-plasma of 68 patients with gastrointestinal cancer, 22 patients with inflammatory bowel disease (IBD) and 60 healthy controls. Sensitivity of Tumor M2-PK was 70.6% for all GI-tumors, that of CA19-9 was 55.4% and that of CEA was 53.3%. In pancreatic cancer CA19-9 showed the best sensitivity. In oesophageal/gastric cancer Tumor M2-PK was most sensitive and in colorectal cancer CEA and Tumor M2-PK showed the best results. The specificity of Tumor M2-PK was 90-96, 7%. In IBD some individuals showed elevated Tumor M2-PK levels but there was no correlation to CRP or to the clinical activity score. The results indicated that Tumor M2-PK might be a valuable marker in gastrointestinal cancer.

Biomarkers, Tumor↗
PubMed1998

Altered growth of transgenic tobacco lacking leaf cytosolic pyruvate kinase.

Previously, we reported that transformation of tobacco (Nicotiana tabacum L.) with a vector containing a potato cytosolic pyruvate kinase (PKc) cDNA generated two plant lines specifically lacking leaf PKc (PKc-) as a result of co-suppression. PKc deficiency in these primary transformants did not appear to alter plant development, although root growth was not examined. Here we report a striking reduction in root growth of homozygous progeny of both PKc- lines throughout development under moderate (600 microE m-2 s-1) or low (100 microE m-2 s-1) light intensities. When both PKc- lines were cultivated under low light, shoot and flower development were also delayed and leaf indentations were apparent. Leaf PK activity in the transformants was significantly decreased at all time points examined, whereas root activities were unaffected. Polypeptides corresponding to PKc were undetectable on immunoblots of PKc- leaf extracts, except in 6-week-old low-light-grown PKc- plants, in which leaf PKc expression appeared to be greatly reduced. The metabolic implications of the kinetic characteristics of partially purified PKc from wild-type tobacco leaves are discussed. Overall, the results suggest that leaf PKc deficiency leads to a perturbation in source-sink relationships.

Cytosol↗
PubMed2005

Effect of exogenous adenosine and monensin on glycolytic flux in isolated perfused normoxic rat hearts: role of pyruvate kinase.

We studied the effect of exogenous adenosine in isolated perfused normoxic rat hearts on glycolytic flux through pyruvate kinase (PK). We compared its effect with that of myxothiazol, an inhibitor of mitochondrial ATP production. Moreover, we tested whether an increase of membrane ionic flux with monensin is linked to a stimulation of glycolytic flux through PK. After a 20-min stabilization period adenosine, myxothiazol or monensin were administrated to the perfusate continuously at various concentrations during 10 min. The contraction was monitored and the lactate production in coronary effluents evaluated. The amount of adenine nucleotides and phosphoenolpyruvate was measured in the frozen hearts. Myxothiazol induced a decrease of the left ventricular developed pressure (LVDP : -40%) together with a stimulation of glycolytic flux secondary to PK activation. In contrast, adenosine primarily reduced heart rate (HR: -30%) with only marginal effects on LVDP. This was associated with an inhibition of glycolysis at the level of PK. The Na+ ionophore monensin affected HR (+14%) and LVDP (+25%). This effect was associated with a stimulation of glycolysis secondary to the stimulation of PK. These results provide new information of action of adenosine in the heart and support the concept of a direct coupling between glycolysis and process regulating sarcolemmal ionic fluxes.

Adenine Nucleotides↗